Normal human cells in culture exhibit limited proliferative potential (cellular senescence). Senescent cells have been found to produce an inhibitor of DNA synthesis that is not made by young cells. Our current hypothesis is that production of this inhibitor of DNA synthesis is the end point of a genetic program that results in cellular senescence. From cell hybrid studies we have determined that this genetic program is dominant and that the phenotype of immortality is recessive. The goal of this proposal is to understand what recessive changes have occurred in immortal cells. Part of the proposal focuses on immortalization of human cells by SV40 virus. From cell hybrid studies we have assigned 22 immortal human cell lines to three complementation groups for immortality. All immortal SV40-transformed cell lines assigned to the same complementation group, indicating that SV40 immortalizes human cells by the same processes. It is clear that the viral genome is not sufficient to maintain immortalization, because human cells containing a stably integrated and expressed viral genome exhibit limited division. Two possibilities remain: (i) The virus or viral gene products interact with some cellular gene to maintain immortalization, or (ii) a cellular gene is modified following viral integration and this gene alone maintains immortalization. In this proposal we will directly test the role of T antigen in the maintenance of immortalization by inactivating the T-antigen-coding sequences in the integrated viral genome of immortal SV40-transformed cells. If T antigen is required to maintain immortality, we will determine with which cellular genes this protein interacts. If T antigen is not required, we will determine whether the viral enhancer is involved in the maintenance of immortalization. The results will clearly demonstrate whether viral sequences are required to maintain immortality and aid in identification of cellular sequences involved. Another aim of this proposal is to determine whether immortal cell lines within any of the complementation groups for immortality are producing the inhibitor of DNA synthesis expressed in senescent cells. Cell lines that are producing the inhibitor will be analyzed to determine whether they have become immortal by expressing genes that act counter to the inhibitor. Cell lines that are not producing the inhibitor will be analyzed to determine whether they have become immortal because of transcriptional or translational block of the inhibitor gene, post- translational modification or inactivation of the inhibitor protein, or modifications in genes that regulate expression of the inhibitor gene.